DNA topoisomerase I (Top1) is ubiquitous and essential in higher eukaryotes. It relieves DNA torsional stress and relaxes DNA supercoiling by introducing DNA single-strand breaks, which are produced by the covalent linking of the Top1 catalytic tyrosine residue (Y723 in humans) to a 3′-DNA phosphate. Thus, these breaks are referred to as “Top1 cleavage complexes”. Once the DNA is relaxed, each break is religated as the 5′-end of the broken DNA reseals the break by attacking the phosphotyrosyl bond, which releases Top1. Top1-DNA cleavage complexes are normally undetectable because they are very transient.
Top1 cleavage complexes can selectively be trapped by the natural alkaloid camptothecin (Hsiang et al., 1985) as the drug binds at the enzyme-DNA interface and prevents DNA religation. Two camptothecin derivatives are used in cancer therapy [topotecan (Hycamtin; GlaxoSmithKline, Uxbridge, Middlesex, UK) and irinotecan (CPT-11, Camptosar; Pfizer, New York, N.Y.)], and several families of non-camptothecin inhibitors are being developed as novel anticancer agents. Top1 cleavage complexes can also be trapped by endogenous DNA lesions, including abasic sites, mismatches, oxidized bases, nicks, and carcinogenic DNA adducts. Hence, DNA modifications such as those associated with oxidative damage (thousands per cell per day) can stabilize Top1 cleavage complexes. In contrast to camptothecins and other Top1 inhibitory drugs, these DNA modifications produce irreversible cleavage complexes when the 5′-end of the DNA is irreversibly misaligned, as in the case of abasic sites or DNA breaks. The irreversible cleavage complexes are commonly referred to as “suicide complexes.” Reversible cleavage complexes trapped by drugs can also be converted into irreversible complexes after collision of replication forks or transcription complexes with the Top1 cleavage complexes.
Tyrosyl-DNA phosphodiesterase (Tdp1) was discovered as an enzyme that specifically removes the 3′-phosphotyrosyl adducts. Top1 needs to be proteolyzed or denatured for Tdp1 to hydrolyze the tyrosyl-DNA bond. Top1 degradation and ubiquitination have indeed been observed after camptothecin treatment.
Tdp1 orthologs are present in all eukaryotic species examined, including yeasts and humans. Sequence comparisons and structural studies revealed that Tdp1 is a member of the phospholipase D (PLD) superfamily, which also includes a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases.
In humans, homozygous mutation in the TDP1 gene (1478A-G) resulting in substitution of histine 493 with arginine is responsible for “spinocerebellar ataxia with axonal neuropathy” (SCAN1). Recent studies demonstrated that SCAN1 cells are hypersensitive to camptothecin and that Tdp1 is required for the repair of abortive Top1 cleavage complexes. Tdp1 forms multiprotein complexes with the single-strand break repair XRCC1 complexes by direct interaction with DNA ligase III. These complexes are catalytically defective in SCAN1 cell extracts, which accumulate Tdp1-DNA intermediates. Tdp1 can also remove glycolate residues from the 3′-end of DNA. 3′-Phosphoglycolate is a common byproduct of DNA double-strand breaks caused by oxidative fragmentation of DNA sugars, which occur as a result of ionizing radiation and oxidative DNA damage. Consistently, extracts from SCAN1 cells are deficient in processing 3′-phosphoglycolate. Thus, Tdp1 seems to repair Top1-DNA adducts and free-radical-mediated DNA breaks. Because the latter can also generate Top1 covalent complexes, Top1 repair is probably a critical function of Tdp1 .
In budding yeast, a T722A mutant Top1 that induces high level of cleavage complexes by increasing their stability results in low viability. However, Tdp1 deficiency alone does not confer hypersensitivity to Top1 cleavage complexes unless an additional mutation of the RAD9 checkpoint gene or the RAD1 endonuclease gene is associated with a TDP1-null mutation. Moreover, Tdp1 overexpression in human cells counteracts DNA damage mediated not only by Top1 but also by Top2. Because cancer cells are characteristically defective in checkpoint and DNA repair, and oncogenic transformation produces high levels of oxidative radicals, it is plausible that Tdp1 inhibitors might be used for anticancer treatment alone or more likely in combination with camptothecins or other Top1 inhibitors.
Just recently, aminoglycosides and other antibiotic ribosome inhibitors were reported as the first pharmacological inhibitors for Tdp1. The only other inhibitors of Tdp1 are vanadate and tungstate, which are general inhibitors of a variety of enzymes involved in phosphoryl transfer reactions. Using recombinant human Tdp1 and model tyrosyl-oligonucleotides substrates, it has been shown that antibiotics that target bacterial ribosomes can inhibit Tdp1 activity. Potential Tdp1 inhibitors are neomycin and tetracycline.
The development of Tdp1 inhibitors as anticancer agents can be envisioned as combinations of Tdp1 and Top1 inhibitors. Tumor cells, whose repair pathways are commonly deficient, might be selectively sensitized to Top1 inhibitors compared with normal cells that contain redundant repair pathways. Moreover, Tdp1 inhibitors might also be effective by themselves as anticancer agents because oncogenic activation tends to increase free radical production and genomic instability. In addition, Tdp1 inhibitors might be valuable as anti-infectious agents because the gene is present in parasites.
Aspects of the present invention employ materials known as supercritical, critical or near-critical fluids. A material becomes a critical fluid at conditions which equal its critical temperature and critical pressure. A material becomes a supercritical fluid at conditions which equal or exceed both its critical temperature and critical pressure. The parameters of critical temperature and critical pressure are intrinsic thermodynamic properties of all sufficiently stable pure compounds and mixtures. Carbon dioxide, for example, becomes a supercritical fluid at conditions which equal or exceed its critical temperature of 31.1° C. and its critical pressure of 72.8 atm (1,070 psig). In the supercritical fluid region, normally gaseous substances such as carbon dioxide become dense phase fluids which have been observed to exhibit greatly enhanced solvating power. At a pressure of 3,000 psig (204 atm) and a temperature of 40° C., carbon dioxide has a density of approximately 0.8 g/cc and behaves much like a nonpolar organic solvent, having a dipole moment of zero Debyes.
A supercritical fluid displays a wide spectrum of solvation power as its density is strongly dependent upon temperature and pressure. Temperature changes of tens of degrees or pressure changes by tens of atmospheres can change a compound solubility in a supercritical fluid by an order of magnitude or more. This feature allows for the fine-tuning of solvation power and the fractionation of mixed solutes. The selectivity of nonpolar supercritical fluid solvents can also be enhanced by addition of compounds known as modifiers (also referred to as entrainers or cosolvents). These modifiers are typically somewhat polar organic solvents such as acetone, ethanol, methanol, methylene chloride or ethyl acetate. Varying the proportion of modifier allows wide latitude in the variation of solvent power.
In addition to their unique solubilization characteristics, supercritical fluids possess other physicochemical properties which add to their attractiveness as solvents. They can exhibit liquid-like density yet still retain gas-like properties of high diffusivity and low viscosity. The latter increases mass transfer rates, significantly reducing processing times. Additionally, the ultra-low surface tension of supercritical fluids allows facile penetration into microporous materials, increasing extraction efficiency and overall yields.
A material at conditions that border its supercritical state will have properties that are similar to those of the substance in the supercritical state. These so-called “near-critical” fluids are also useful for the practice of this invention. For the purposes of this invention, a near-critical fluid is defined as a fluid which is (a) at a temperature between its critical temperature (Tc) and 75% of its critical temperature and at a pressure at least 75% of its critical pressure, or (b) at a pressure between its critical pressure (Pc) and 75% of its critical pressure and at a temperature at least 75% of its critical temperature. In this definition, pressure and temperature are defined on absolute scales, e.g., Kelvin and psia. To simplify the terminology, materials which are utilized under conditions which are supercritical, near-critical, or exactly at their critical point will jointly be referred to as “SCCNC” fluids or referred to as “SFS.”
SCCNC fluids can be used for the co-encapsulation of the hydrophobic Top1 inhibitor (camptothecins) and the hydrophilic Tdp1 inhibitor (antibiotics) in phospholipid nanosomes (small, uniform liposomes). Camptothecins are quite hydrophobic and will be packaged in the lipid bilayer. Antibiotics such as tetracycline and neomycin are quite water-soluble and will be packaged in the aqueous core of phospholipid nanosomes.
The nanosomal formulation of the co-encapsulated drugs will result in reduced systemic toxicity, due to the masking of the cytotoxic effects of camptothecins and Tdp1 inhibitors. Additionally, the stability of the lactone ring in the nanosomes will be improved as a result of protection from the neutral pH of the blood stream. By increasing residence time in the circulatory system, the nanosomes increase therapeutic efficacy of the combination drugs. Optionally, pegylated phospholipids will be utilized to provide steric hindrance that will further increase residence time and therapeutic efficacy as is done with Doxil® liposome encapsulated doxorubicin. Furthermore, phospholipids linked with specific antibodies or ligands will be utilized to target the co-encapsulated camptothecin and Tdp1 inhibitor to specific cancers in the colon, lung or ovary. Such smart targeting will further reduce toxicities associated with both Top1 and Tdp1 inhibitors while increasing efficacy and therapeutic index.
At present, there are no available technologies that can readily co-encapsulate hydrophobic and hydrophilic drugs in phospholipid nanosomes in a single step, scalable process. Conventional processes for the encapsulation of hydrophobic drugs utilize many processing steps and require large quantities of organic solvents. These processes are very time consuming, costly and inefficient. Generally, such phospholipid liposomes have a wide dispersion of particle size. Such phospholipid liposomes tend to have a median size greater than 100 microns in diameter. In addition, the exposure of therapeutic agent to the organic solvent may adversely affect the integrity of the final product. Other conventional processes for the encapsulation of hydrophilic drugs into phospholipid liposomes utilize high pressure homogenization that requires a significant amount of recycling, generates heat with every pass through the homogenizer, and could be contaminated with heavy metal particles. These conventional processing methods may also compromise sterility, or do not provide sterility.
Embodiments of the present invention address these problems inherent in the prior art with the application of supercritical, critical or near-critical fluids with or without a cosolvent or modifier.